(Adapted from the applicant's abstract) The overall goal of this research program is to develop improved, clinically applicable transduction and transplantation protocols that result in the efficient repopulation of recipients with genetically modified hematopoietic cells capable of expressing the inserted genes. In light of the current inability to demonstrate efficient gene transfer into hematopoietic stem cells of large animals or patients, we will focus our efforts on testing several hypotheses regarding the genetic modification of stem cells using retroviral vectors that have not yet been adequately examined. In addition, we will study in detail the ability of different retroviral vectors to provide for the expression of inserted genes in hematopoietic cells in vivo. Assessment of the efficiency of gene transfer and extent of gene expression achieved using different transduction protocols will be made through a series of parallel studies involving murine and human cells. Our methods of analysis will include the use of murine bone marrow transplantation models and the use of in vitro colony assays for human hematopoietic progenitors. A small number of candidate protocols will be further evaluated in autologous and MHC-matched allogeneic bone marrow transplantation models in the pig. The specific aims of the research program are: 1) To test the hypothesis that the titer and specific host range of recombinant retroviruses are critical determinants of the ability to efficiently transduce hematopoietic stem cells; 2) To test the hypothesis that the use of specific purified populations of hematopoietic stem cells are recipients for gene transfer will lead to the improved representation of genetically modified hematopoietic cells in recipients reconstituted with genetically modified cells; 3) To test the hypothesis that the isolation of transduced stem cells prior to transplantation can be used to improve the proportion of genetically modified cells detectable after transplantation; 4) To further characterize the expression potential of specific retroviral vectors in mice reconstituted with cells transduced using the optimal protocols developed in specific aims 1-3, 5) To use autologous and MHC-matched allogeneic bone marrow transplantation models in the pig to further validate strategies for transduction and/or transplantation of stem cells that have been developed on the basis of the studies in specific aims 1-3.